Ozone exposed epithelial cells modify cocultured natural killer cells - PubMed (original) (raw)
Ozone exposed epithelial cells modify cocultured natural killer cells
Loretta Müller et al. Am J Physiol Lung Cell Mol Physiol. 2013.
Abstract
Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs.
Figures
Fig. 1.
Nasal epithelial cells (NECs) and natural killer (NK) cell interactions in vivo are mimicked by NEC-NK cocultures in vitro. A: flow cytometric analysis of superficial nasal scrape biopsies. The biopsies were digested to obtain a single cell suspension and analyzed for the presence of NK cells (CD45+CD56+CD3−). Representative scatterplot is shown. B: hematoxylin- and eosin-stained paraffin-embedded sections of superficial nasal scrape biopsies. The image shows ciliated NECs above multiple layers of basal ECs and an apically located lymphocytic cell (white arrowhead). C: in vitro coculture model of differentiated human NECs and human peripheral blood NK cells (arrow) added on the apical side. NK cells are located on the apical side of NECs and are in contact with the luminal border of the NECs. D: laser-scanning microscopy image of a NEC-NK cell coculture. NK cells (green) were labeled with a fluorescein-emitting dye before addition to the NECs. The F-actin cytoskeleton of the NECs (red) was stained with phalloidin-rhodamine and the cell nuclei of both cell types (blue) with DAPI. En face view demonstrates the presence of several NK cells located apically on the NEC monolayer. Cross-sectional view of the cocultures shows NK cells in direct contact with NECs and partially enclosed in the NEC monolayer (white arrowhead).
Fig. 2.
Flow cytometry analysis of NK cells cocultured with air- or O3-exposed NECs. A: NK cell surface markers. B: intracellular mediator expression as mean fluorescence intensity fold induction of O3 over air of the entire population. C: percent interferon-γ (IFN-γ)- and IL-4-positive NK cells. Data are presented as fold induction of the mean fluorescence intensity (MFI) (A and B) or of percentage of positive cells (C) ± SE of cells exposed to O3 over cells exposed to air (n = 5–6), *P < 0.05.
Fig. 3.
Flow cytometry data of NK cells incubated with conditioned media from air- and O3-exposed NECs. A: surface markers. B: intracellular markers. Data are presented as fold induction of the mean fluorescence intensity ± SE of cells exposed to O3 over air (n = 8), *P < 0.05.
Fig. 4.
Flow cytometry analysis of NEC surface makers. Analysis of MICA/B, ULBP3, and CXCR3 in NECs alone (A) or NECs cocultured with NK cells (B). Data are presented as fold induction of mean fluorescence intensity ± SE of cells exposed to O3 over air (n = 9), *P < 0.05 and **P < 0.01.
Fig. 5.
Cytotoxic potential of NK cells was reduced after coculturing with O3-exposed NECs. The data represent the percentage of lysed target cells as a measure of NK cell cytotoxic potential. *P < 0.05.
Fig. 6.
Analysis of the role of ULBP3 and MICA/B in the effects on NK cells. A: effect of stimulation with recombinant ULBP3 (10 ng for 4 h) on intracellular IFN-γ and IL-4 of NK cells. B and C: effect of blocking ULBP3 and MICA/B after exposing NECs to air or O3 on IFN-γ (B) and IL-4 (C) in NK cells cocultured with NECs. Data are presented as fold induction of mean fluorescence intensity (MFI) of cells exposed to O3 over air (A, n = 6). *P < 0.05.
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